JP2625033B2 - Composition for inducing granulocyte production or B cell production in peripheral blood - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to the use of polypeptides called cartilage-inducing factors (CIFs), and functional as factors for inducing proliferation of B cells or granulocytes in peripheral blood. And a plurality of polypeptides related to.

BACKGROUND OF THE INVENTION U.S. Pat. No. 4,806,523, filed by the same applicant, describes CIF from two bovine bones named CIF-A and CIF-B. The two CIFs have a molecular weight of about 26,000 daltons by SDS-PAGE and are dimers.
This CIF in itself exhibits chondrogenic activity in vitro, as measured by the production of cartilage-specific proteoglycans (PG) in an agarose gel medium model using rat fetal mesenchymal cells . However,
Neither of the two CIFs has their own chondrogenic activity in vivo. The amino acid sequence of CIF-A has a portion of the N-terminal sequence (30 amino acids) corresponding to the amino acid sequence reported in a polypeptide derived from human placenta called β-type transforming growth factor (TGF-β) It has been shown. The N-terminal sequence portion of CIF-B is different from that of TGF-β. These two CIFs are TGF
Shows activity (ability to induce substrate-independent growth of normal rat kidney cell colonies in soft agar) in -β assay.

TGF-β from bovine kidney, human placenta and human platelets
Are described in International Patent Application No.PCT / US83 / 01460 published March 29, 1984 as WO 84/01106, and EPA 8445016.5 published December 01, 1984 as 0128849. ing. These applications are based on such TGF
Provides data indicating that -β (1) promotes cell proliferation in the soft agar culture assay described above and (2) promotes cell proliferation and protein accumulation in a rat soft tissue wound healing model. doing. These applications show that TGF-β has a molecular weight of about 26,00 by SDS-PAGE.
Identified as a 0 Dalton dimer. Currently, CIF-A
It is called TGF-β1, and CIF-B is called TGF-β2.

DISCLOSURE OF THE INVENTION The present invention is based on the discovery that CIF (ie, TGF-β) induces the production of B cells in peripheral blood or induces the production of granulocytes. For convenience, the term CIF is used herein and in the claims as a general term encompassing CIF-A, CIF-B, TGF-β and functional equivalents thereof.

Accordingly, the present invention relates to the use of CIF in the manufacture of a medicament for inducing the production of B cells or inducing the production of granulocytes in peripheral blood.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is the amino acid sequence of platelet-derived human TGF-β monomer.

FIG. 2 shows the fraction obtained by gel filtration in Example 1. 3 is a graph of the optical density (absorbance) (280 nm) of FIG.

FIG. 3 shows elution fractions obtained by ion-exchange chromatography for separation in Example 1. 3 is a graph of the optical density (280 nm) of FIG.

FIG. 4 graphically illustrates the increase in peripheral blood leukocytes following TGF-β1 administration in vivo, as reported in the Examples.

FIGS. 5A and 5B graphically illustrate the increase in peripheral blood B cells following TGF-β1 administration in vivo, as reported in the Examples.

6A and 6B graphically illustrate the increase in the percentage of both small and large B cells after TGF-β1 administration in vivo, as reported in the Examples.

MODE FOR CARRYING OUT THE INVENTION The term "functional equivalent" for describing a polypeptide
Are polypeptides having the same amino acid sequence as the polypeptide referred to, whether natural or synthetic, and regardless of species or origin, and are substantially homologous (ie, at least 90% identical in amino acid sequence). Yes, polypeptides in which different amino acid sequences do not adversely affect biological activity (ie, the ability to reduce B cell and granulocyte proliferation).

CIF-A, CIF-B and TGF-β are available from Methods for Prepara
tion of media, Supplements, and Substrate for Serum-
Free Animal Cell Culture (1984) pp181-194, Alan RL
Shows activity in the TGF-β assay described by iss, Inc. This assay measures the ability to induce substrate-independent growth in non-neoplastic normal rat kidney (NRK) fibroblasts by measuring the formation of cell colonies in soft agar. TG from platelets, placenta and kidney tissue
Methods for obtaining F-β are described in International Patent Publication No. WO 84/01106 and EPA 128,849. In summary, those methods involve extracting raw materials with acid-ethanol,
Size extraction of the extract by gel filtration and high-performance liquid chromatography (HPLC)
Isolating TGF-β.

A method for isolating CIF from bovine bone is described in commonly assigned US Pat. No. 4,806,523. This method uses an extractant that solubilizes non-fibrous proteins (eg, 4M
Demineralized bone (DM with guanidine hydrochloride, 8M urea)
Extracting B), gel-filtrating the extract to obtain a fraction smaller than 30 kDa, chromatography of the fraction on carboxymethylcellulose (CMC) at pH 4.5-5.5, preferably 4.8, C
A step of eluting the fraction adsorbed on the MC with a concentration gradient of NaCl, and a step of purifying the protein from a portion eluted with about 150-250 mM NaCl by reverse phase HPLC or gel electrophoresis.

To date, CIF-A, C isolated from natural sources
IF-B and TGF-β are dimers of polypeptides with a molecular weight of about 25-26 kDa, as measured by SDS-PAGE. Nature (1985) 316 : 701-705 reports the nucleotide sequence of the cDNA and expresses human TGF-β from platelets.
The amino acids are estimated. Humans from mature platelets
TGF-β has been identified as a homodimer of 112 amino acid long monomers.

TGF-β and CIF-A from platelets / placenta / kidney and
CIF-B is not species-specific when it comes to TGF-β activity. Thus, the polypeptides are highly conserved among animal species (ie, certain polypeptides from different mammals may differ slightly in that one or more amino acid residues are added, deleted or substituted). However, it has an amino acid sequence that does not adversely affect the activity of a molecule that is not species-specific), and has the function of interspecific hybridization. Therefore, CIF-A, CIF-B and
TGF-β can be derived from cells or tissues of various animals or obtained by recombinant DNA technology. Correlated to that, a vertebrate CIF (TGF-β)
Can be used to treat other vertebrates. The most common use of CIF (TGF-β) as an anti-inflammatory agent is in the treatment of humans; livestock such as cattle, sheep and pigs; and sports or pet animals such as dogs, cats and horses. . CI
FA and CIF-B are preferably used in the method of the present invention.

Examples The following examples illustrate particular embodiments of the present invention. The present invention is not limited by this.

Example 1 (Preparation of CIF from bone) A. Preparation of desalted bone Bovine metatarsal bone was freshly obtained from a slaughterhouse and transported on dry ice. The bone was cleaned except for bone marrow and non-bone tissue, cut into pieces less than 1 cm in diameter and powdered at 4 ° C. using a mill. Powdered bone is 9.4 liters per kg of bone
Wash twice with double-distilled water for about 15 minutes each, and then
Washed overnight at 4 ° C. with 01N HCl. The washed bone
Degreasing was performed three times with three volumes of ethanol and then three times with three volumes of diethyl ether, and each was washed for 20 minutes, all at room temperature. Next, the defatted bone powder obtained is
Desalting was performed at 4 ° C. in 0.5 N HCl (25 L / kg of defatted bone). The acid was decanted, the resulting DMB was washed until the pH after washing was greater than 4, and the DNB was dried by suction filtration.

B. Extraction of non-collagenous proteins 3.3 g of DMB prepared in 4 M guanidine hydrochloride per kg, pH 6.
8, 10 mM ethylenediaminetetraacetic acid (EDTA), 1 mM P
MSF, extracted with 10 mM NEM for 16 hours, suction filtered the suspension,
The insoluble material was extracted again for 4 hours. The soluble fractions are combined, concentrated at least 5-fold by ultrafiltration using an Amicon ultrafiltration device (10K) and the concentrate is
The dialysis was performed with 6 changes of 35 volumes of cold deionized water and lyophilized. All operations in this paragraph, except for lyophilization performed under standard lyophilization conditions,
Performed at 4 ° C.

C. Gel filtration Was re-dissolved in 4M guanidine hydrochloride, 10m in 4M guanidine hydrochloride, 0.02% sodium azide, pH 6.8
Fractionation was performed on a column of Sephacryl® S-200 equilibrated in M EDTA. Fractions were analyzed by absorbance at 280 nm and fractions were collected as shown in FIG. The fraction F2 of FIG. 1 which constitutes a fraction of proteins of low molecular weight (LMW, 10,000-30,000 daltons) was dialyzed by changing 180 times volume of deionized water six times and freeze-dried. Lyophilization and dialysis (4
All operations except (° C) were performed at room temperature.

D. Ion exchange chromatography Fraction F2 in 6 M urea, 10 mM NaCl, 1 mM NEM, pH
Dissolve in 50 mM sodium acetate at 4.8 and add 5 at 10,000 rpm.
Centrifuged for minutes. The supernatant was fractionated on CM52 (commercially available CMC) equilibrated with the same buffer. Bound protein was eluted from the column using a gradient of 10 mM to 400 mM NaCl in the same buffer. At this time, the total volume
At 350 mL, the flow rate was 27 mL / hr. CM-1, CM-2 and CM-3
The three main fractions, named, were collected as shown in FIG. CM-2 and CM-3 contain approximately 150-250 mM Na
Eluted with Cl. Each fraction was dialyzed for 4 days with 6 changes of 110 volumes of deionized water and lyophilized. All the above operations were performed at room temperature except for dialysis (4 ° C.).

E. Reverse phase HPLC Lyophilized Fractions CM-2 and CM-3
Were each dissolved in 0.1% trifluoroacetic acid (TFA), a portion of the solution was loaded on a Vydac® C18 reverse phase HPLC column (4.6 mm ID × 25 cm), and the flow rate was changed with 0.1% TFA for 5 minutes. Washing was performed at 1 mL / min. The elution solvent was a concentration gradient of 0% -60% acetonitrile in 0.1% TFA at a rate of 2% / min.

Reverse phase HPLC of the collected CM-2 and CM-3 yielded two peaks. Peak A is about 29.5 minutes and Peak B is about
31.2 minutes. The proteins at these peaks are according to the invention of US Patent Application No. 630,938 and are named CIF-A and CIF-B, respectively.

The protein was stored at −20 ° C. in a 0.1% TFA / acetonitrile elution solution until use.

F. Characterization of CIF-A and CIF-B Table 1 below shows the partial amino acid composition of CIF-A and CIF-B.

According to SDS-PAGE analysis of CIF-A and CIF-B, both were approximately
It can be seen that it has a molecular weight of 26,000 daltons. Both proteins show activity in the TGF-β assay described above compared to those reported for TGF-β from human platelets, human placenta or bovine kidney.

Analysis of the amino acid sequence of the first 30 amino acids at the N-terminus of CIF-A and CIF-B revealed the following as a result.

The N-terminal amino acid sequence of CIF-A is human TGF-
Same as reported for β (see Nature above).

Example 2 (Increasing peripheral blood B cells) This experiment demonstrates the induction of B cells in peripheral blood using CIF-A.

TGF-β1 (CIF-A) was purified from bovine bone and 45% EtO
H, concentrated in 11 mM HCl to a concentration of about 5000 μg / mL and stored at −20 ° C. until needed. Next, TGF-β1 was replaced with PBS.
(0.1% MSA) to a concentration of 125 μg / mL and 1% E
Neutral pH was established with tOH.

C57B1 / 6 male mice were treated with TGF-β1 (25 μl) for 14 days.
g, 0.2 mL, subcutaneously). Control mice received vehicle without TGF-β1.
Twenty-four hours after the last treatment, mice were evaluated for histological evaluation of spleen and bone marrow, hematological evaluation, body weight, and phenotype of leukocytes, spleen and bone marrow from blood. Leukocytes were classified using a monoclonal antibody and a fluorescence activated cell sorter (FACS). B cells are MA
b Classified using B220 (ATCC No. TIB 164). Granulocytes were obtained from Dr. RLCoffman (DNAX Research Institute, Palo
Alto, CA). T _H
Cells were sorted with anti-CD4 MAb and TC _{/ S} cells with anti-CD8. Note that both anti-CD4 MAb and anti-CD8
Obtained from Becton-Dickinson. Propidium iodide staining was used to determine the percentage of non-viable cells.

Hematological evaluation shows that treatment with TGF-β1 significantly increases the total number of white blood cells in peripheral blood (FIG. 4) and decreases the total number of red blood cells. The increase in white blood cells
It was determined by FACS analysis to be at least in part due to an increase in B cells (the proportion of B220 ⁺ cells doubled). FIG. 5A shows the analysis by FACS of the control, and FIG. 5B shows the FGF-β1
The analysis by ACS is shown. Which T in peripheral blood
There were no changes in cell markers. Although the proportion of granulocytes (8C5 ⁺ ) is decreasing, the decrease is largely due to an increase in B cells rather than a decrease in the absolute number of granulocytes. Particle size analysis of B220 ⁺ (forward light scattering) showed that the proportion of both small and large B cells (ie B220 ⁺ ) was increasing. FIG. 6A shows the particle size analysis of the control, and FIG. 6B shows the particle size analysis of the sample treated with TGF-β1.

8C5 staining recorded increased granulocytes in the spleen and bone marrow. B cells and T cells in spleen and bone marrow between control and treated mice
No difference was seen in the percentage of cells. Furthermore, TGF-β
1 treatment did not affect propidium iodide staining, indicating that administration of TGF-β1 did not affect leukocyte survival.

Thus, TGF-β1 increased the number of B cells in peripheral blood. Thus, formulations of TGF-β may be used, for example, in irradiated bone marrow transplant recipients, patients treated with chemotherapy, and B cell proliferation or function birth defects, B cell number reduction or humoral immunity. Useful for treatment of related indications.

Modifications of the form for practicing the present invention will be obvious to those skilled in the fields of protein chemistry, immunology, pharmacology and related fields and are within the scope of the appended claims.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Ellingsworth, Larry United States of America 95148 San Jose, Woodley Drive 3566 (56) Reference The Journal of Immunology, Vol. 140, No. 8 (1988) p. 2661-2665

Claims (1)

(57) [Claims] (1) inducing an increase in the number of B cells in peripheral blood;
Alternatively, CI used to induce the production of granulocytes
A composition containing F (TGF-β).